A large plasmid (Ti-plasmid), present in nature in tumour provoking strains of Agrobacterium tumefaciens, is responsible for tumour induction in dicotyledonous plants (Van Larebeke et al., Nature (London) 252, 169-170 (1974)l Zaenen et al., F. Mol. Biol 86, 109-127 (1974)). A specific part of the Ti-plasmid, called T-region, is transferred by the bacterium to the plant cell and is integrated into the core DNA (Chilton et al., Cell 11, 263-271 (1977)).
The integrated DNA is expressed (Willmitzer et al., Mol. Gen. Genet. 182, 255-262 (1981)) and this expression forms the molecular basis of the plant disease Crown Gall. Resulting tumour cells, in contradistinction to normal plant cells, can be grown on synthetic media without adding the plant hormones auxine and cytokinine. (UBraum, Proc. Natl. Acad. Sci 44, 344:349 (1958)). The tumour cells also contain specific compounds, called opines, which are not present in normal cells, the genetic information of which lies coded on the transferred T-DNA (Bomhoff et al., Mol. Gen. Genet. 145, 177:181 (1976); Schroder et al., FEBS Lett. 129, 166-168 (1981)).
Besides the T-region, which is found in the plant cell, the Ti-plasmid contains a second region which is essential for the virulence qualities of the bacterium (Omms et al., J. Bacteriol. 144, 82-91 (1980; Garfinkel et al., J. Bacteriol. 144, 732-743 (1980)). This region was never seen in tumour cells and genetic analyses have shown that mutations in this area (leading to much weakened virulence or complete avirulence) are trans complementable by wild types of genes, which are present on clones or R Prime Plasmids (Hille et al. Plasmid 7, 197-118 (1982); Klee et al., J. Bacteriol. 150, 327-331)). Seven genetic loci have been determined in this Vir (Virulence) region, called Vir A, B, C, D, E, F and G (Klee et al., J. Bacteriol. 150, 327-331 (1982); Hille et al., Plasmid 7, 107-118 (1982); Hooykaas et al., in press (1984)).
The Vir region and the T-region can be physically separated from one another on two compatible plasmids without any negative effect on the capacity of agrobacteria to incorporate the T-region or artificial T-region into the plant cell (Hoekema et al., Nature (London), 303, 179-180 (1983)). A binary vector system based on this invention can be used for the genetic manipulation of plant cells. (Described in Dutch patent specification no. 83 00698 and European patent application no. 842002396, and in Dutch patent application 84 01048). In Dutch patent application 84 01048 we reported that monocotyledonous species, which was thought not to be susceptible to Agrobacterium, was indeed able to respond to Agrobacterium. After wounding the plants and infecting with Agrobacterium it appeared from the experiments that (1) cells of monocotyledonous plants were positively transformed by A. tumefaciens; (2) plant material isolated from non-wounded or wounded but not infected monocotyledonous plants contained neither octopine nor nopaline. Opines were not discovered in plant material isolated from plants infected with avirulent strains while plant cells obtained from wound sites infected with virulent A. tumefaciens strains appeared to nearly always contain opines. Consequently, it was concluded that the Ti plasmid is suitable as a vector for monocotyledonous plant cells.